Abstract 1995 Characterization of Diadenosine Polyphosphatases of the Nudix Hydrolase Superfamily in M. tuberculosis and M. leprae

M. tuberculosis and M. leprae contain diadenosine polyphosphatase mRNA decapping Nudix hydrolases, and we are characterizing these enzymes as potential novel antibiotic targets. In M. tuberculosis there is the primary Nudix ApnAase and the secondary Nudix ApnAase, and there are orthologs of these two enzymes in M. leprae. The diadenosine polyphosphatases from Legionella pneumophilia and Bartonella bacilliformis are considered "invasion enzymes"; when the genes for these enzymes are knocked out, these bacteria are less able to invade their host cells. It is of interest to know whether these enzymes have the same or similar function in M. tuberculosis and M. leprae. If they are found to be involved in invasiveness and thus in virulence, then these enzymes could be novel antibiotic targets. We have cloned and overexpressed each protein and have subcloned each protein into a HisTag vector to optimize purification. The M. leprae enzymes express too insolubly to purify and characterize, and thus we are working on increasing the expression of soluble protein so that we can study these enzymes as well; currently, we know that they each have ApnAase activity. We have tried to increase enzyme solubility by changing the overexpression conditions including lower temperatures, less IPTG, the addition of GroESL, the use of Rosetta E. coli, and the addition of diglycine. This research has been supported by an NIH AREA grant, a CUR-Goldwater Scholars Faculty Mentor Award, an ASBMB undergraduate research award, and a RIT honors SURF.